Alpha 1-6 fucosyltransferase

ABSTRACT

An isolated, porcine α1-6 fucosyltransferase having the following physico-chemical properties:  
     (1) action: transferring fucose from guanosine diphosphate-fucose to a hydroxy group at 6-position of GluNAc closest to R of a receptor (GlcNAcβ1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlucNAc-R wherein R is an asparagine residue or a peptide chain carrying said residue, whereby to form (GlcNAcβ1-2Manα1-6)-(GlcNAcβ1-2Manα1-3)Manα1-4GlcNAcβ1-4(Fucα1-6)GlucNAc-R  
     (2) optimum pH: about 7.0  
     (3) pH stability: retains activity after 5 hours of treatment at 4° C. at a pH range of 4.0-10.0  
     (4) optimum temperature: about 30-37° C.  
     (5) inhibition or activation: no requirement for divalent metal for expression of activity; no inhibition of activity in the presence of 5 mM EDTA  
     (6) molecular weight: about 60,000 by SDS-polyacrylamide gel electrophoresis  
     is provided. An isolated polynucleotide encoding porcine α1-6 fucosyltransferase is also provided. Expression vectors comprising a polynucleotide encoding porcine α1-6 fucosyltransferase are also provided.

TECHNICAL FIELD

[0001] The present invention relates to an α1-6 fucosyltransferasederived from pig or human. More particularly, the present inventionrelates to a novel α1-6 fucosyltransferase derived from human, which isan enzyme that transfers fucose from guanosine diphosphate (GDP)-fucoseby α1→6 linkage to N-acetylglucosamine (GlcNAc) bound to Asn at the stemof asparagine type sugar chain (Asn type sugar chain) and which isuseful in the field of glyco-technology for modification and synthesisof sugar chain and/or for the diagnosis of diseases such as malignanttumor, and to a gene encoding said enzyme.

BACKGROUND ART

[0002] The structure and function of sugar chain moiety of complexcarbohydrates, such as glycoprotein and glycolipid, derived from higherorganisms have been drawing much attention in recent years, and manystudies are under way. While a sugar chain is formed by the action ofglycohydrolase and glycosyltransferase, glycosyltransferase contributesgreatly to its formation.

[0003] Using a sugar nucleotide as a sugar donor, glycosyltransferasetransfers a sugar to a receptor sugar chain, thereby to elongate thesugar chain. The specificity for the structure of receptor sugar chainis stringent, such that one glycoside linkage is formed by thecorresponding one transferase. Hence, glycosyltransferases are used forstructural studies of sugar moiety of complex carbohydrate, forfacilitated synthesis of a particular sugar chain structure, and formodification of native sugar chain structure.

[0004] Besides, glycosyltransferases are expected to be usable for themodification of the nature of complex carbohydrate and cells, by meansof artificial alteration of sugar chain. For this end, the developmentof various glycosyltransferases having identified substrate specificityhas been awaited.

[0005] An α1-6 fucosyltransferase is an important enzyme found in Golgiappratus of organelle, which is considered to be one of the enzymes thatcontrol processing of asparagine-linked sugar chain. Therefore, theenzyme will be useful for the elucidation of control mechanism andcontrol of formation of sugar chain structure, once acted on anasparagine-linked sugar chain.

[0006] In addition, the activity of α1-6 fucosyltransferase and theproportion of reaction products of this enzyme are known to increase incertain diseases such as liver cancer and cystic fibrosis. Therefore, arapid development of the method for diagnosis of these diseases has beendesired, which involves determination of the activity of this enzyme,Northern blot using a cDNA encoding α1-6 fucosyltransferase, or RT-PCRassay of mRNA amount transcribed and expressed in the living body.

[0007] The activity of α1-6 fucosyltransferases has been detected inbody fluids or organs of various animals and culture cells thereof, andthere has been known, as a purified enzyme product, an enzyme derivedfrom human cystic fibrosis cell homogenates [Journal of BiologicalChemistry, vol. 266, pp. 21572-21577 (1991)]. According to this report,however, the enzyme is associated with drawbacks in that (1) its optimumpH is 5.6 which is different from physiological pH, (2) it hasrelatively low molecular weights (34,000 and 39,000) bySDS-polyacrylamide gel electrophoresis, (3) its large scale and stablesupply is practically unattainable due to its being derived from humancell, and others.

[0008] This enzyme is obtained as a membrane-bound enzyme, and requiresbovine serum for culturing the cells, which in turn results in difficultpurification of the enzyme and a huge amount of money necessary forculture of the cells to be a starting material. Consequently, stablesupply of this enzyme preparation is all but impractical.

[0009] While a chemical synthesis is often employed for synthesizing asugar chain, the synthesis of oligosaccharides requires many steps thathave been necessitated by its complicated synthesis route andspecificity of the reaction, so that it involves various practicalproblems. Particularly, binding of fucose to GlcNAc bound to Asn ofasparagine-linked sugar chain by α1→6 linkage is extremely difficult dueto the instability of fucose.

DISCLOSURE OF THE INVENTION

[0010] It is therefore an object of the present invention to stablyprovide an α1-6 fucosyltransferase in large amounts, which is useful asa reagent for structural analysis of sugar chain or glyco-technology, oras diagnostics.

[0011] Another object of the present invention is to provide a method ofproducing α1-6 fucosyltransferase in large amounts by the use of ahuman- or porcine-derived α1-6 fucosyltransferase gene. It is aimed touse such specific genes so as to enable development of a method fordiagnosis of diseases by Northern blot using a DNA encoding said enzyme,or by RT-PCR assay of mRNA amount transcribed and expressed in theliving body.

[0012] In an attempt to achieve the above-mentioned objects, the presentinventors started the study of an enzyme capable of linking fucose toGlcNAc linked to Asn of asparagine type sugar chain by α1→6 linkage,using a fluorescence-labeled substrate analogous to an asparagine typesugar chain which is a receptor of this enzyme. As a result, they havefound the activity of this enzyme in the extract fractions of porcinebrain which is readily available as a starting material to be purified,and they have purified said enzyme from said fractions and elucidatedthe enzymatic and physico-chemical properties, which resulted in thecompletion of the invention.

[0013] Accordingly, the present invention relates to a porcine-derivedα1-6 fucosyltransferase having the following physico-chemical properties(hereinafter this enzyme is referred to as porcine α1-6fucosyltransferase).

[0014] (1) Action: transferring fucose from guanosine diphosphate-fucoseto the hydroxy group at 6-position of GluNAc closest to R of a receptor(GlcNAcβ1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlucNAc-R whereinR is an asparagine residue or a peptide chain carrying said residue,whereby to form(GlcNAcβ1-2Manα1-6)-(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlucNAc-R.

[0015] In the above formula, asparagine residue at R is a residuewherein the acid amide group at the side chain of asparagine is bound tothe hydroxy group at the anomer position of the reducing terminal ofsugar chain, and a peptide chain having said residue is a peptide chainhaving said residue in the peptide to which two or more amino acids arebound, which is preferably a peptide chain having -Asn-(X)-Ser/Thr-.

[0016] (2) optimum pH: about 7.0

[0017] (3) pH stability: stable in the pH range of 4.0-10.0 by treatmentat 4° C. for 5 hours

[0018] (4) optimum temperature: about 30-37° C.

[0019] (5) inhibition or activation: no requirement for divalent metalion for expression of activity; no inhibition of activity even in thepresence of 5 mM EDTA

[0020] (6) molecular weight: about 60,000 by SDS-polyacrylamide gelelectrophoresis.

[0021] The present inventors have purified α1-6 fucosyltransferase alonefrom porcine brain, analyzed the amino acid sequence of this protein andcloned a gene based on the partial amino acid sequence to accomplish thepresent invention.

[0022] That is, the present invention provides a gene encoding porcineα1-6 fucosyltransferase.

[0023] The present invention also provides an expression vectorcontaining a gene encoding porcine α1-6 fucosyltransferase.

[0024] The present invention further provides a transformant cellobtained by transforming a host cell with an expression vectorcontaining a gene encoding porcine α1-6 fucosyltransferase.

[0025] The present invention yet provides a method for producing arecombinant α1-6 fucosyltransferase, comprising culturing a transformantcell obtained by transforming a host cell with an expression vectorcontaining a gene encoding porcine α1-6 fucosyltransferase, andharvesting the α1-6 fucosyltransferase from the culture thereof.

[0026] The present inventors have reached the present invention bypurifying protein having an α1-6 fucosyltransferase activity from humancell culture broth and elucidating its enzymatic property.

[0027] Accordingly, the present invention relates to an α1-6fucosyltransferase derived from human, having the followingphysico-chemical property (hereinafter this enzyme is to be referred toas human α1-6 fucosyltransferase).

[0028] (1) Action: transferring fucose from guanosine diphosphate-fucoseto the hydroxy group at 6-position of GluNAc closest to R of a receptor(GlcNAcβ1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlucNAc-R whereinR is an asparagine residue or a peptide chain carrying said residue,whereby to form(GlcNAcβ1-2Manα1-6)-(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlucNAc-R.

[0029] In the above formula, asparagine residue at R is a residuewherein the acid amide group at the side chain of asparagine is bound tothe reducing terminal hydroxy group of sugar chain, and a peptide chainhaving said residue is a peptide chain having said residue in thepeptide to which two or more amino acids are bound, which is preferablya peptide chain having -Asn-(X)-Ser/Thr-.

[0030] (2) optimum pH: about 7.5

[0031] (3) pH stability: stable in the pH range of 4.0-10.0 by treatmentat 4° C. for 5 hours

[0032] (4) optimum temperature: about 30-37° C.

[0033] (5) inhibition or activation: no requirement for divalent metalion for expression of activity; no inhibition of activity even in thepresence of 5 mM EDTA

[0034] (6) molecular weight: about 60,000 by SDS-polyacrylamide gelelectrophoresis.

[0035] The present inventors have purified α1-6 fucosyltransferase alonefrom human culture cell, analyzed the amino acid sequence of thisprotein and cloned a gene based on the partial amino acid sequence toaccomplish the present invention.

[0036] That is, the present invention provides a gene encoding humanα1-6 fucosyltransferase.

[0037] The present invention also provides an expression vectorcontaining a gene encoding human α1-6 fucosyltransferase.

[0038] The present invention further provides a transformant cellobtained by transforming a host cell with an expression vectorcontaining a gene encoding human α1-6 fucosyltransferase.

[0039] The present invention yet provides a method for producing arecombinant α1-6 fucosyltransferase, comprising culturing a transformantcell obtained by transforming a host cell with an expression vectorcontaining a gene encoding human α1-6 fucosyltransferase, and harvestingthe α1-6 fucosyltransferase from the culture thereof.

[0040] The starting material for the purification of the enzyme of thepresent invention is, for example, the organ and body fluid of pighaving α1-6 fucosyltransferase activity. Examples of the organ includebrain, spermary, pancreas, lung, kidney and the like. The body fluid ofpig such as blood and sera can be also used.

[0041] The porcine α1-6 fucosyltransferase of the present invention canbe obtained by preparing a crude extract containing the enzyme from, forexample, homogenates of porcine brain and separating the enzyme fromthis extract. In this case, since α1-6 fucosyltransferase in the porcinebrain is a membrane-bound enzyme, a crude extract solution containingthe enzyme is generally obtained from brain lysate using a suitablesurfactant. This extract undergoes various known purification steps togive a purified enzyme product. The purification may include, forexample, concentration using an ultrafiltration membrane, desalting,affinity column chromatography wherein a substrate analog isimmobilized, ion exchange column chromatography, hydrophobic columnchromatography and the like in suitable combination to give asubstantially homogeneous enzyme product which is free of contaminantproteins such as other transferases. For example, porcine brain isdisrupted in a Waring blender in a phosphate buffer and membranefractions are collected by ultracentrifugation. The objective enzyme isextracted with a phosphate buffer containing a surfactant (TritonX-100), and the supernatants are collected by ultracentrifugation togive a crude extract containing the enzyme. By applying affinity columnchromatography using a guanosine diphosphate(GDP)-hexanolamine-sepharose, aGlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-asparagine-sepharoseand the like, the fractions showing fucosyltransferase activity arecollected and purified.

[0042] The physico-chemical property of α1-6 fucosyltransferase derivedfrom porcine brain, which is one aspect of the present invention, is asfollows.

[0043] (1) Action: transferring fucose from guanosine diphosphate-fucoseto the hydroxy group at 6-position of GluNAc closest to R of a receptor(GlcNAcβ1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlucNAc-R whereinR is an asparagine residue or a peptide chain carrying said residue,whereby to form(GlcNAcβ1-2Manα1-6)-(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlucNAc-R.

[0044] (2) Determination of activity:

[0045] The activity of the porcine α1-6 fucosyltransferase wasdetermined as follows. That is, a compound of the following formula,wherein the sugar chain end asparagine was fluorescence-labeled with4-(2-pyridylamino)butylamine [PABA: —NH₂(CH₂)₄—NH-pyridine] was used asa substrate for determination of enzyme activity:

[0046] wherein PA means pyridylamino. By the use of this substrate, theproduct from the enzyme reaction, wherein fucose has been transferred byα1→6 linkage, can be assayed by detecting fluorescence by highperformance liquid chromatography.

[0047] Specifically, the determination includes the following steps. Asample solution (10 μl) and 1.25% Triton X-100 are added to a 250 mM MESbuffer containing 62.5 μM of fluorescence-labeled receptor substrate ofthe above formula and 625 μM of a donor substrate (GDP-fucose), pH 7.0,40 μl, and mixed. The mixture is reacted at 37° C. for one hour, andboiled for 5 minutes to stop the reaction. The reaction mixture issubjected to high performance liquid chromatography and the peak of thereaction product is assayed with a fluorescence detector. One unit ofthe enzyme amount corresponds to the amount capable of forming 1 pmoleof GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc-R(wherein R is Asn-NH—(CH₂)₄—NH-pyridine) in one minute under theseconditions.

[0048] (3) Optimum pH:

[0049] The α1-6 fucosyltransferase derived from porcine brain(hereinafter this enzyme is referred to as porcine brain α1-6fucosyltransferase) shows a high activity at nearly pH 7.0-7.5.

[0050] (4) pH Stability:

[0051] The porcine brain α1-6 fucosyltransferase is relatively stable atpH 4-10, and more stable at pH 5-9.

[0052] (5) Optimum Temperature:

[0053] The porcine brain α1-6 fucosyltransferase has an optimumtemperature at nearly 37° C. and retains sufficient activity at 20-40°C.

[0054] (6) Divalent Metal Ion Requirement:

[0055] The porcine brain α1-6 fucosyltransferase shows sufficientactivity even in the absence of divalent metal ion, such as magnesium,manganese and the like. It also shows sufficient activity even in thepresence of 5 mM EDTA, which is a chelating agent.

[0056] (7) Molecular Weight:

[0057] A purified product of the porcine brain α1-6 fucosyltransferaseshows a single band at a molecular weight of about 60,000 bySDS-polyacrylamide gel electrophoresis.

[0058] Judging from the above properties, the porcine brain α1-6fucosyltransferase is a novel enzyme apparently different fromconventionally known α1-6 fucosyltransferase derived from human cysticfibrosis cells (optimum pH 5.6, molecular weights 34,000 and 39,000), interms of optimum pH, metal ion requirement and molecular weight.

[0059] The inventive porcine α1-6 fucosyltransferase is expected to beextremely useful for (1) synthesis of sugar chain compounds whereinsugar chain compounds having 1-6 fucose are synthesized using the enzymeof the present invention, (2) modification of sugar chain structure andfunctional analysis wherein a fucose is newly introduced into asparaginetype sugar chain to artificially modify the sugar chain structure,whereby changes in cell function and control mechanism of the processingof complex carbohydrate, as well as the role of sugar chain, can beelucidated, (3) diagnosis of lesions based on enzyme activity whereindiseases such as cancer can be diagnosed by determining the activity ofthe enzyme of the present invention which reflects various lesionscaused by tumorigenic transformation, (4) diagnosis of various diseaseswherein a specific antibody against the enzyme of the present inventionis prepared and used for the diagnosis, and the like.

[0060] Another aspect of the present invention is a gene encodingporcine α1-6 fucosyltransferase, which includes a gene encoding α1-6fucosyltransferase and including a gene encoding amino acid sequencedepicted in Sequence Listing, SEQ ID NO:2. A different embodimentthereof is a gene encoding α1-6 fucosyltransferase and includingnucleotide sequence depicted in Sequence Listing, SEQ ID NO:1.

[0061] One aspect of the present invention is a gene encoding porcineα1-6 fucosyltransferase and including a gene encoding an amino acidsequence resulting from substitution, insertion, deletion or additionwith respect to at least one amino acid of the amino acid sequencedepicted in Sequence Listing, SEQ ID NO:2.

[0062] Another aspect of the present invention is a gene encodingporcine α1-6 fucosyltransferase and including a nucleotide sequenceresulting from substitution, insertion, deletion or addition withrespect to at least one nucleotide of the nucleotide sequence depictedin Sequence Listing, SEQ ID NO:1.

[0063] The present invention also includes, as one aspect thereof, agene that hybridizes to at least a part of a gene encoding porcine α1-6fucosyltransferase and including nucleotide sequence depicted inSequence Listing, SEQ ID NO:1.

[0064] The expression vector of the present invention contains a geneencoding the above-mentioned porcine α1-6 fucosyltransferase.

[0065] The transformant host cell of the present invention has beentransformed with the above-mentioned expression vector.

[0066] The host cell is exemplified by microorganisms, such asEscherichia coli, yeast, bacterial cells and the like. It also includesanimal cells such as insect cells, COS-1 cells, CHO cells and the like,and plant cells, such as tobacco cells, Arabidopsis cells and the like.

[0067] The vector may be any which is selected according to the host tobe transformed. In the case of Escherichia coli, for example, pUC19 maybe used; in the case of yeast, pYEUra3™ may be used; in the case ofinsect cells, pBLUE Bac4 may be used; in the case of COS-1 cells, pSVK3may be used; and in the case of tobacco cells and Arabidopsis cells, pBImay be used.

[0068] The method for preparing the inventive recombinant α1-6fucosyltransferase includes culturing the above-mentioned transformantcells and harvesting α1-6 fucosyltransferase from the culture.

[0069] According to the present invention, α1-6 fucosyltransferase aloneis purified from porcine brain, and subjected to amino acid analysis ofthis protein. Its partial amino acid sequence is determined and a primerfor PCR is prepared based on the amino acid sequence. Using this primer,PCR is performed using cDNAs derived from porcine brain as a template toamplify a gene encoding α1-6 fucosyltransferase to give a probe. Thisprobe is used to screen clones containing cDNA encoding α1-6fucosyltransferase, from the cDNA library derived from porcine brain.The cDNA encoding α1-6 fucosyltransferase is isolated and used toexpress α1-6 fucosyltransferase.

[0070] To be specific, the purified porcine α1-6 fucosyltransferase isused to analyze amino acid sequences. For example, SDS-polyacrylamidegel electrophoresis is applied, after which the protein is transferredto PVDF membrane by electroblotting, and the PVDF membrane containingca. 60 kDa band is cut out and subjected to sequencing using a proteinsequencer. As a result, the amino acid sequence of the amino terminal ofα1-6 fucosyltransferase depicted in Sequence Listing, SEQ ID NO:3 isobtained.

[0071] Separately, purified α1-6 fucosyltransferase is subjected toSDS-polyacrylamide gel electrophoresis and the peptide fragmentsseparated by electrophoresis are transferred to PVDF membrane byelectroblotting. Then, the PVDF membrane containing 60 kDa band is cutout and lysed on said PVDF membrane, using, for example, a protease suchas lysylendopeptidase. The lysate is extracted from the sections of saidPVDF membrane, and the extract is subjected to reversed phase highperformance liquid chromatography to separate the lysate.

[0072] Then, using the amino acid sequences, a mixed primer for PCR isprepared. For example, a mixed primer having a nucleotide sequencedepicted in SEQ ID NO:7 is synthesized from the amino acid sequencedepicted in SEQ ID NO:3, and a mixed primer having a nucleotide sequencedepicted in SEQ ID NO:8 is synthesized from the amino acid sequencedepicted in SEQ ID NO:4, respectively using a DNA synthesizer, and usedfor the screening of cDNA of α1-6 fucosyltransferase.

[0073] For example, 25 cycles of PCR are performed to amplify DNAfragments of ca. 1.45 kbp, using cDNA from porcine brain as a templateand mixed primers of SEQ ID NO:7 and SEQ ID NO:8, wherein PCR at 94° C.(1 min), 55° C. (2 min) and 72° C. (3 min) is one cycle.

[0074] Then, using the amplified DNA fragments as a probe, clonescontaining cDNA encoding α1-6 fucosyltransferase are screened from thecDNA library derived from porcine brain by a plaque hybridizationmethod. The cDNA encoding α1-6 fucosyltransferase can be isolated fromthe obtained clones. The nucleotide sequence of the obtained cDNA andthe amino acid sequence deduced from said nucleotide sequence are shownin SEQ ID NO:1 and SEQ ID NO:2.

[0075] Said cDNA is subeloned into an expression vector such as pSVK3.The host cells, such as COS-1 cells, transformed with said subclonedplasmid, are cultured and α1-6 fucosyltransferase is obtained from theculture.

[0076] In the present invention, the above-mentioned transformant cellsare cultured and α1-6 fucosyltransferase is harvested from the culture,whereby recombinant α1-6 fucosyltransferase is obtained. The method forharvesting the enzyme from the culture is a conventional one.

[0077] The gene encoding the porcine α1-6 fucosyltransferase of thepresent invention and DNA fragments (which are the lysates thereof) maybe used for the detection of the expression of α1-6 fucosyltransferasein the living body, and thus are useful for the genetic diagnosis ofcertain diseases such as liver cancer and cystic fibrosis.

[0078] In addition, the polypeptide that is encoded by these genes canbe used to immunologically prepare various antibodies which are usefulfor diagnosis and purification of α1-6 fucosyltransferase.

[0079] The starting material for the purification of the enzyme in thisinvention may be any as long as it is a human cell culture mediumexhibiting α1-6 fucosyltransferase activity. For example, humanpancreatic cancer cells, human gastric cancer cells, human myeloma tumorcells and the like may be used as the cells having α1-6fucosyltransferase activity.

[0080] While the human α1-6 fucosyltransferase is present in the cellmembrane as a membrane-bound enzyme, it is cleaved by protease at a siteunaffecting the enzyme activity and released into the culture medium asa soluble enzyme. Thus, the culture medium can be used as a crude enzymesolution, without complicated steps such as disruption of cells andsolubilizing of the enzyme. Besides, the use of cells capable of growthin serum-free media enables economical production of a crude enzymesolution having a high purity. The culture medium is concentrated anddesalted, and subjected to ion exchange chromatography, affinitychromatography and the like to give a purified enzyme product free ofcontaminant transferases and glycosidase activity.

[0081] α1-6 Fucosyltransferase is purified from human gastric cancercells by, for example, culturing human gastric cancer cell MKN45 withoutserum and purifying the enzyme from the obtained culture medium. In thiscase, α1-6 fucosyltransferase of human gastric cancer cell MKN45 iscleaved by protease in the cells at a site unaffecting the enzymeactivity and released into culture medium as a soluble α1-6fucosyltransferase. Therefore, the culture medium can be used as a crudeenzyme solution, without complicated steps such as disruption of cellsand solubilizing of the enzyme with a surfactant. The crude enzymesolution is subjected to known purification steps to give a purifiedenzyme product.

[0082] In the present invention, a serum-free culture medium of humangastric cancer cell MKN45 is concentrated by filtration through anultrafiltration membrane, and then the buffer is changed to a Tris-HClbuffer containing 5 mM 2-mercaptoethanol and 0.1% CHAPS[3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate], pH 7.5, togive a crude enzyme solution.

[0083] This enzyme solution is subjected to column chromatography usingQ-sepharose, GDP-hexanolamine-sepharose,(GlcNAcβ1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manβ1-4GlucNAcβ1-4GlucNAc-asparagine-sepharoseand the like to collect active fractions, from which thefucosyltransferase of the present invention can be purified.

[0084] The physico-chemical property of human α1-6 fucosyltransferase ofthe present invention is as follows.

[0085] (1) Action: transferring fucose from guanosine diphosphate-fucoseto the hydroxy group at 6-position of GluNAc closest to R of a receptor(GlcNAcβ1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manα1-4GlcNAcβ1-4GlucNAc-R whereinR is an asparagine residue or a peptide chain carrying said residue,whereby to form(GlcNAcβ1-2Manα1-6)-(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlucNAc-R.

[0086] (2) Determination of Enzyme Activity:

[0087] The activity of the human α1-6 fucosyltransferase was determinedas follows. That is, a compound of the above-mentioned formula, whereinthe asparagine on the end of sugar chain was fluorescence-labeled with4-(2-pyridylamino)butylamine [PABA: —NH₂(CH₂)₄—NH-pyridine], was used asa substrate for determination of enzyme activity. By the use of thissubstrate, the product from the enzyme reaction, wherein fucose istransferred by α1→6 linkage, can be assayed by detecting fluorescence byhigh performance liquid chromatography.

[0088] Specifically, the determination included the following steps. Anenzyme solution (10 μl) was added to a 250 mM MES buffer containing 62.5μM of fluorescence-labeled receptor substrate of the above formula and625 μM of a donor substrate (GDP-fucose), pH 7.0, 40 μl, and mixed. Themixture was reacted at 37° C. for one hour, and boiled for 5 minutes tostop the reaction. The reaction mixture is subjected to high performanceliquid chromatography and the peak of the reaction product is assayedwith a fluorescence detector.

[0089] One unit of the enzyme amount corresponded to the amount capableof producing 1 pmole ofGlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)-Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc-R(wherein R is Asn-NH—(CH₂)₄—NH-Pyridine) in one minute under theseconditions.

[0090] (3) Optimum pH:

[0091] The human α1-6 fucosyltransferase shows high activity at nearlypH 7.0-7.5, as shown by a curve in FIG. 1. In FIG. 1, the determinationwas performed using 500 mM MES buffer (black circle) at pH 4.5-7.5 and100 mM Tris-HCl buffer (white circle) at pH 7.0-9.0.

[0092] (4) pH Stability:

[0093] The human α1-6 fucosyltransferase is stable at about pH 4-10,particularly at pH 5-9, as shown in FIG. 2. The buffers used for thedetermination were 50 mM acetate buffer (black triangle) at pH 3.5-5.5,50 mM MES buffer (black circle) at pH 5.5-7.5, 50 mM Tris-HCl buffer(white circle) at pH 7.5-9.0, and sodium hydrogencarbonate buffer (whitetriangle) at pH 9.0-11.5. The enzyme of the present invention wastreated in each buffer at each pH at 4° C. for 5 hours, and the residualactivity was determined. FIG. 1 is a graph showing the relationshipbetween pH (axis of abscissa) and relative activity (%, axis ofordinate) of the human α1-6 fucosyltransferase obtained by the presentinvention, and FIG. 2 is a graph showing pH (axis of abscissa) andresidual activity (%, axis of ordinate).

[0094] (5) Optimum Temperature:

[0095] The human α1-6 fucosyltransferase has an optimum temperature atnearly 37° C. as shown in FIG. 3 and is usable at 20-40° C. A frozenproduct thereof is stable at −20° C. for at least several months.

[0096] (6) Divalent Metal Ion Requirement:

[0097] Many glycosyltransferases require divalent metal ion for theiractivity, such as magnesium, manganese and the like. This human α1-6fucosyltransferase shows sufficient activity in the absence of divalentmetal ion or in the presence of EDTA, which is a chelating agent, anddoes not require divalent metal ion.

[0098] (7) Molecular Weight:

[0099] A purified product of the human α1-6 fucosyltransferase of thepresent invention shows a single band at a molecular weight of about60,000 by SDS-polyacrylamide gel electrophoresis.

[0100] (8) Morphology:

[0101] While the human α1-6 fucosyltransferase is intrinsically presentin cell membrane as a membrane-bound enzyme, it is cleaved by proteasein the cultured cell at a site unaffecting the enzyme activity andreleased into a culture medium as a soluble enzyme permitting easyhandling, unlike porcine-derived α1-6 fucosyltransferase and α1-6fucosyltransferase derived from human cystic fibrosis cells heretoforereported.

[0102] Judging from the above properties, the human α1-6fucosyl-transferase is a novel enzyme apparently different fromconventionally known α1-6 fucosyltransferase derived from human cysticfibrosis cells (optimum pH 6.5, molecular weights 34,000 and 39,000), interms of optimum pH, metal requirement and molecular weight.

[0103] The human α1-6 fucosyltransferase is used for the followingpurposes.

[0104] (1) Artificial modification of sugar chain structure byintroducing fucose anew into the asparagine-linked sugar chain, wherebycell apparatus and control mechanism of processing of sugar chain ofcomplex carbohydrate, as well as the role of sugar chain, can beelucidated.

[0105] (2) Diagnosis of various diseases based on the activity of theinventive enzyme.

[0106] (3) Diagnosis of various diseases wherein a specific antibodyagainst the enzyme of the present invention is prepared and used for thediagnosis.

[0107] The present invention is a gene encoding human α1-6fucosyl-transferase, which includes, as one embodiment, a gene encodingα1-6 fucosyltransferase and including a gene encoding an amino acidsequence depicted in Sequence Listing, SEQ ID NO:10. A differentembodiment thereof is a gene encoding α1-6 fucosyltransferase inclusiveof nucleotide sequence depicted in Sequence Listing, SEQ ID NO:9. Afurther aspect of the present invention is a gene encoding α1-6fucosyltransferase and including a nucleotide sequence from 198thadenine to 1919th guanine as depicted in Sequence Listing, SEQ ID NO:9.

[0108] One aspect of the present invention is a gene encoding α1-6fucosyltransferase and including a gene encoding an amino acid sequenceresulting from substitution, insertion, deletion or addition withrespect to at least one amino acid of the amino acid sequence depictedin Sequence Listing, SEQ ID NO:10.

[0109] Another aspect of the present invention is a gene encoding α1-6fucosyltransferase and including a nucleotide sequence resulting fromsubstitution, insertion, deletion or addition with respect to at leastone nucleotide of the nucleotide sequence depicted in Sequence Listing,SEQ ID NO:9.

[0110] The present invention also includes, as one embodiment, a genewhich hybridizes to at least a part of gene encoding α1-6fucosyltransferase and including nucleotide sequence depicted inSequence Listing, SEQ ID NO:9.

[0111] The expression vector of the present invention contains a geneencoding the above-mentioned α1-6 fucosyltransferase.

[0112] The transformant host cell of the present invention has beentransformed with the above-mentioned expression vector.

[0113] The host cell is exemplified by microorganisms, such asEscherichia coli, yeast, bacterial cells and the like. It also includesanimal cells such as insect cells, COS-1 cells, CHO cells and the like,and plant cells, such as tobacco cells, Arabidopsis cells and the like.

[0114] The vector may be any which is selected according to the host tobe transformed. In the case of Escherichia coli, for example, pUC19 maybe used; in the case of yeast, pYEUra3 T may be used; in the case ofinsect cells, pBLUE Bac4 may be used; in the case of COS-1 cells, pSVK3may be used; and in the case of tobacco cells and Arabidopsis cells, pBImay be used.

[0115] The method for preparing the recombinant α1-6 fucosyltransferaseincludes culturing the above-mentioned transformant cells and harvestingα1-6 fucosyltransferase from the culture.

[0116] According to the present invention, α1-6 fucosyltransferase aloneis purified from human gastric cancer cells, and subjected to amino acidanalysis of this protein. Its partial amino acid sequence is determinedand a primer for PCR is prepared based on the amino acid sequence. Usingthis primer, PCR is performed using cDNAs derived from human gastriccancer cells as a template to amplify a gene encoding α1-6fucosyltransferase to give a probe. This probe is used to screen clonescontaining cDNA encoding α1-6 fucosyltransferase, from the cDNA libraryderived from human gastric cancer cells. The cDNA encoding α1-6fucosyltransferase is isolated and used to express α1-6fucosyltransferase.

[0117] To be specific, the purified α1-6 fucosyltransferase is used toanalyze amino acid sequence. For example, it is subjected toSDS-polyacrylamide gel electrophoresis, after which the protein istransferred to PVDF membrane by electroblotting, and the PVDF membranecontaining ca. 60 kDa band is cut out and subjected to sequencing by aprotein sequencer. As a result, the amino acid sequence of the aminoterminal of α1-6 fucosyltransferase depicted in Sequence Listing, SEQ IDNO:11 is obtained.

[0118] Separately, purified α1-6 fucosyltransferase is subjected toSDS-polyacrylamide gel electrophoresis, along with a protease such aslysylendopeptidase, and the peptide fragments separated byelectrophoresis are transferred to PVDF membrane by electroblotting.Then, the band containing the peptide fragments is cut out and subjectedto sequencing with a protein sequencer. Thus, partial amino acidsequences of α1-6 fucosyltransferase as depicted in Sequence Listing,SEQ ID NO:12 and SEQ ID NO:13 are obtained. Then, using these amino acidsequences, a mixed primer for PCR is prepared. For example, a mixedprimer having a nucleotide sequence depicted in SEQ ID NO:14 issynthesized from the amino acid sequence depicted in SEQ ID NO:12, and amixed primer having a nucleotide sequence depicted in SEQ ID NO:15 issynthesized from the amino acid sequence depicted in SEQ ID NO:13,respectively using a DNA synthesizer, and used for the screening of cDNAof α1-6 fucosyltransferase.

[0119] For example, 36 cycles of PCR are performed to amplify the DNAfragments of ca. 200 bp, using cDNA from human gastric cancer cells as atemplate and mixed primers of SEQ ID NO:14 and SEQ ID NO:15, wherein PCRat 94° C. (30 sec), 46° C. (30 sec) and 72° C. (1.5 min) is one cycle.

[0120] Then, using the amplified DNA fragments as a probe, clonescontaining cDNA encoding α1-6 fucosyltransferase are screened from thecDNA library derived from human gastric cancer cells by a plaquehybridization method. The cDNA encoding α1-6 fucosyltransferase can beisolated from the obtained clones. The nucleotide sequence of theobtained cDNA and the amino acid sequences deduced from said nucleotidesequence are shown in SEQ ID NO:9 and SEQ ID NO:10.

[0121] Said cDNA is subcloned into an expression vector such as pSVK3.The host cells such as COS-1 cells transformed with said subclonedplasmid are cultured and α1-6 fucosyltransferase is obtained from theculture.

[0122] In the present invention, the above-mentioned transformant cellsare cultured and α1-6 fucosyltransferase is harvested from the culture,whereby a recombinant α1-6 fucosyltransferase is obtained.

[0123] The method for harvesting the enzyme from the culture is aconventional one.

[0124] The gene encoding the human α1-6 fucosyltransferase of thepresent invention and DNA fragments (which are the lysates thereof) maybe used for the determination of the expression of α1-6fucosyltransferase in the living body and thus are useful for geneticdiagnosis of certain diseases such as liver cancer and cystic fibrosis.

[0125] In addition, the polypeptide that is encoded by these genes canbe used to immunologically prepare various antibodies which are usefulfor diagnosis and purification of α1-6 fucosyltransferase.

BRIEF DESCRIPTION OF THE DRAWINGS

[0126]FIG. 1 shows optimum pH of the porcine brain α1-6fucosyl-transferase of the present invention.

[0127]FIG. 2 shows pH stability of the porcine brain α1-6fucosyl-transferase of the present invention.

[0128]FIG. 3 shows optimum temperature of the porcine brain α1-6fucosyltransferase of the present invention.

[0129]FIG. 4 shows optimum pH of the human α1-6 fucosyltransferase ofthe present invention.

[0130]FIG. 5 shows pH stability of the human α1-6 fucosyltransferase ofthe present invention.

[0131]FIG. 6 shows optimum temperature of the human α1-6fucosyl-transferase of the present invention.

EMBODIMENT OF THE INVENTION

[0132] The present invention is described in more detail by way ofExamples.

[0133] In the present invention, the enzyme activity is determined asfollows.

[0134] A compound of the following formula, wherein the asparagine onthe end of sugar chain had been fluorescence-labeled with4-(2-pyridyl-amino)butylamine [PABA: —NH(CH₂)₄—NH-pyridine] was used asa substrate for the determination of enzyme activity. By the use of thissubstrate, the product from the enzyme reaction wherein fucose has beentransferred by α1→6 linkage can be assayed by detecting the fluorescenceby high performance liquid chromatography.

[0135] Specifically, the determination includes the following steps. Asample solution (10 μl) and 1.25% Triton X-100 are added to a 250 mM MESbuffer containing 62.5 μM of fluorescence-labeled receptor substrate ofthe above formula and 625 μM of a donor substrate (GDP-fucose), pH 7.0,40 μl, and mixed. The mixture is reacted at 37° C. for one hour, andboiled for 5 minutes to stop the reaction. The reaction mixture issubjected to high performance liquid chromatography and the peak of thereaction product is assayed with a fluorescence detector. One unit ofthe enzyme amount corresponds to the amount capable of producing 1 pmoleofGlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)-Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc-R(wherein R is Asn-NH—(CH₂)₄—NH-pyridine) in one minute under theseconditions.

EXAMPLE 1

[0136] (1) Preparation of Porcine Brain Lysate and Crude ExtractSolution

[0137] Porcine brain (100 g) was disrupted in a Waring blender in a 20mM potassium phosphate buffer (pH 7.0) and membrane fractions werecollected by ultracentrifugation. The membrane fractions were extractedwith the same buffer containing Triton X-100 (concentration 0.5%) toextract the enzyme. After the extraction, the supernatants werecollected by centrifugation to give an extract containing a crudeenzyme.

[0138] (2) Purification of Enzyme from Crude Extract Solution

[0139] AGlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-asparagine-sepharosecolumn (column of asialoagalactoglyco-peptide derived from transferrin)was equilibrated with a 20 mM potassium phosphate buffer (pH 7.0)containing 0.05% Triton X-100 and 50 mM KCl, and the crude extractsolution prepared in (1) above was applied. The column was washed withsaid buffer until the protein was not detected in the unadsorbedfractions. The active fractions were eluted with the same buffercontaining 1M KCl. Then, the active fractions of the enzyme wereconcentrated using an ultrafiltration membrane and desalted, and appliedto a GDP-hexanolamine-sepharose column equilibrated with the samebuffer. The elution was performed using the same buffer containing 100mM GDP. Then, the active fractions were collected and concentrated usingan ultrafiltration membrane, and desalted to give porcine brain α1-6fucosyltransferase. The porcine brain α1-6 fucosyltransferase thusobtained showed a single band at a molecular weight of about 60,000 bySDS-polyacrylamide gel electrophoresis. No other bands ascribed toimpurities were found and the enzyme was free of other transferaseactivities, thus indicating that the enzyme obtained was highlypurified.

[0140] The optimum pH (determined by changing the pH of buffer) of theenzyme of the present invention is shown in FIG. 1. The enzyme showedhigh activity at around pH 7.0-7.5. The buffer used was 200 mM MESbuffer (black circle). In this graph, the axis of abscissa shows pH ofα1-6 fucosyltransferase obtained in the present invention and the axisof ordinate shows relative activity (%).

[0141] The pH stability of the enzyme of the present invention wasexamined in the same manner. FIG. 2 shows residual activity aftertreating the enzyme in each buffer at each pH, 4° C. for 5 hours. Theenzyme was comparatively stable at about pH 4-10, and particularlystable at pH 5-9. The buffers used were 50 mM acetate buffer (blacktriangle) at pH 3.5-5.5, 50 mM MES buffer (black circle) at pH 5.5-7.5,50 mM Tris-HCl buffer (white circle) at pH 7.5-9.0, and sodiumhydrogencarbonate buffer (white triangle) at pH 9.0-11.5. The axis ofabscissa of the graph shows pH of α1-6 fucosyltransferase obtained inthe present invention and the axis of ordinate shows residual activity(%).

[0142] As shown in FIG. 3, the optimum temperature of the enzyme of thepresent invention was found to be at about 37° C. and the enzyme wasconsidered to retain sufficient activity in the range of 20-40° C. Afrozen product thereof was stable at −20° C. for at least severalmonths. The buffer used was 200 mM MES buffer (black circle), pH 7.0.The axis of abscissa of the graph shows treatment temperature (° C.) andthe axis of ordinate shows relative activity (%) of the α1-6fucosyltransferase obtained in the present invention.

[0143] While many glycosyltransferases require divalent metal ion fortheir activity, such as magnesium, manganese and the like, the enzymeshowed sufficient activity in the absence of such divalent metal ion.Inasmuch as it showed sufficient activity even in the presence of 5 mMEDTA, which is a chelating agent, it is concluded that the enzyme doesnot require a divalent metal ion.

EXAMPLE 2

[0144] Determination of Amino Terminal Amino Acid Sequence of PorcineBrain α1-6 Fucosyltransferase

[0145] Purified porcine brain α1-6 fucosyltransferase (5 μg) wassubjected to SDS-polyacrylamide gel electrophoresis, after which theprotein was transferred to PVDF membrane (Millipore) by electroblotting.The PVDF membrane was stained with Coomassie Brilliant Blue G250, and asingle band of porcine brain α1-6 fucosyltransferase was detected at 60kDa.

[0146] Then, the PVDF membrane containing said band was cut out, and,after destaining with 50% methanol, subjected to Biosystem 473A proteinsequencer (Applied Biosystems) to determine amino terminal amino acidsequence of α1-6 fucosyltransferase. The amino acid sequence determinedis depicted in Sequence Listing, SEQ ID NO:3.

EXAMPLE 3

[0147] Determination of Partial Amino Acid Sequence of Porcine Brainα1-6 Fucosyltransferase

[0148] Purified porcine brain α1-6 fucosyltransferase (13 μg) wassubjected to SDS-polyacrylamide gel electrophoresis, after which theprotein was transferred to PVDF membrane (Millipore) by electroblotting.The PVDF membrane was stained with Coomassie Brilliant Blue G250, and asingle band of porcine brain α1-6 fucosyltransferase was detected at 60kDa.

[0149] Then, the PVDF membrane containing said band was cut out anddestained with 50% methanol. Said PVDF membrane section was treated in100 mM Tris-HCl buffer-5% acetonitrile (pH 8.2) containing 1 μg oflysylendopeptidase, at 37° C. for 12 hours for proteolysis. The PVDFmembrane section which underwent proteolysis was ultrasonicated toextract the proteolysis product. The proteolysis product thus obtainedwas separated by a reversed phase high performance liquid chromatographyusing a C-18 column to give 3 peptide fragments. The substancecontaining said peptide fragments, which was separated by the reversedphase high performance liquid chromatography, was applied topolybrene-coated precycled glass fiber filter activated withtrifluoroacetate and dried, and then subjected to Biosystem 473A proteinsequencer (Applied Biosystems) to determine partial amino acid sequenceof porcine brain α1-6 fucosyltransferase. The determined amino acidsequence is depicted in Sequence Listing, SEQ ID NOs:4-6.

EXAMPLE 4

[0150] Preparation of Probe DNA by PCR

[0151] Mixed primers shown in SEQ ID NO:7 and SEQ ID NO:8 weresynthesized from the amino acid sequences obtained in Examples 2 and 3.The mixed primer shown in SEQ ID NO:7 was used as a sense primer, andthe mixed primer shown in SEQ ID NO:8 was used as an antisense primerfor PCR. To be specific, 25 cycles of PCR were performed wherein PCR at94° C. (1 min), 55° C. (2 min) and 72° C. (3 min) using 2 μg of porcinebrain-derived cDNA, 25 pmole of sense primer (mixed primer shown in SEQID NO: 7), 25 pmole of antisense primer (mixed primer shown in SEQ IDNO:8) and a reaction mixture (50 μl) of 50 mM potassium chloride-10 mMTris-HCl buffer (pH 8.3)-1.5 mM magnesium chloride-0.001% gelatin-200 μMdNTP, containing 2.5 units of Taq DNA polymerase was one cycle.

[0152] The reaction mixture (10 μl) after PCR was subjected to 0.7%agarose gel electrophoresis to confirm the PCR reaction product DNAfragments. As a result of PCR performed using a mixed primer shown inSEQ ID NO:7 and a mixed primer shown in SEQ ID NO:8 in combination, a1.45 kbp DNA fragment was confirmed by agarose gel electrophoresis.

[0153] This DNA fragment was suboloned into plasmid pT7BLUEt-Vector(Novagen) and nucleotide sequence was confirmed. As a result, a DNAcorresponding to the amino acid sequence depicted in Sequence Listing,SEQ ID NOs:3-6 was detected, whereby the DNA fragment was confirmed tobe a part of α1-6 fucosyltransferase gene.

EXAMPLE 5

[0154] Isolation of Porcine Brain α1-6 Fucosyltransferase Gene

[0155] The DNA fragments obtained in Example 4 were labeled with α-³²PdCTP (3000 Ci/mmol, Amersham) and used as a probe to screen clonescontaining cDNA encoding α1-6 fucosyltransferase, from porcinebrain-derived λ gt11 cDNA library (Clonetech) by plaque hybridizationmethod.

[0156] As a result of screening of about 400,000 plaques, 5 positiveclones c1, c2, c3, c4 and c5 were obtained. Said clones c1 and c2 werepostulated to contain a full length α1-6 fucosyltransferase gene in viewof their length. Thus, the nucleotide sequences of c1 and c2 weredetermined, and a nucleotide sequence depicted in SEQ ID NO:1 wasobtained.

EXAMPLE 6

[0157] Expression of Porcine Brain α1-6 Fucosyltransferase Gene

[0158] The coding region of α1-6 fucosyltransferase gene was subclonedinto expression vector pSVK3 from clones containing cDNA encodingporcine brain α1-6 fucosyltransferase obtained in Example 5. Theexpression vector containing said α1-6 fucosyltransferase gene wasintroduced into COS-1 cells. After 48 hours of incubation, culture cellswere collected and the cells were disrupted. The enzyme activity of α1-6fucosyltransferase in the obtained lysate was determined.

[0159] As a control, the enzyme activity of α1-6 fucosyltransferase inthe lysate of COS-1 cells, into which mock pSVK3 had been introduced,was determined. As a result, the control hardly showed activity, whereasCOS-cells into which the expression vector containing said α1-6fucosyltransferase gene had been introduced, showed a high activity of2360 nmole/h/mg protein.

EXAMPLE 7

[0160] (1) Preparation of Crude Enzyme Solution from Serum-Free CultureMedium of Human Gastric Cancer Cell MKN45

[0161] Human gastric cancer cell MKN45 was cultured in a serum-freemedium (RPMI1640 medium:Ham's F-12 medium=1:1) containing sodiumselenite and canamycin, at 37° C. in 5% CO₂. The resulting serum-freeculture medium (100 l) was concentrated to 2 l by ultrafiltration. Thebuffer was changed to a Tris-HCl buffer containing 5 mM2-mercaptoethanol and 0.1% CHAPS[3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate], pH 7.5, togive a crude enzyme solution. This crude enzyme solution was subjectedto column chromatography using Q-sepharose, GDP-hexanolamine-sepharose,(GlcNAcβ1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-asparagine-sepharoseand the like to collect active fractions, from which the human α1-6fucosyltransferase of the present invention could be purified.

[0162] (2) Preparation of Enzyme

[0163] The crude enzyme solution obtained in (1) above was subjected tothe following purification steps. That is, the solution was applied to aQ-sepharose column equilibrated with Tris-HCl buffer containing 5 mM2-mercaptoethanol and 0.1% CHAPS, pH 7.5. The column was washed with a5-fold amount of the same buffer and the active fractions eluted withthe same buffer containing 0.1 M NaCl were collected. The activefractions were concentrated using an ultrafiltration membrane and thebuffer was changed to Tris-HCl buffer containing 5 mM 2-mercaptoethanoland 0.7% CHAPS, pH 7.5, after which the fractions were applied toGDP-hexanolamine-sepharose column equilibrated with the same buffer. Theelution was performed by the linear gradient of NaCl from 0 M to 0.5 M.

[0164] The active fractions from 0.15 M to 0.3 M were collected andconcentrated using an ultrafiltration membrane. After desalting, thefractions were applied to a(GlcNAcβ1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-asparagine-sepharosecolumn equilibrated with Tris-HCl buffer containing 5 mM2-mercaptoethanol and 0.7% CHAPS, pH 7.5. The elution was performed bythe linear gradient of NaCl from 0 M to 0.5 M.

[0165] The active fractions from 0.2 M to 0.5 M were collected andconcentrated using an ultrafiltration membrane. Desalting gave humanα1-6 fucosyltransferase.

[0166] The thus-obtained human α1-6 fucosyltransferase fractions showeda single band at a molecular weight of about 60,000 bySDS-polyacrylamide gel electrophoresis. No other activities, such asthose of transferase and glycosidase, were found and this purifiedenzyme was sufficiently usable as a reagent for sugar chain studies.

[0167] The optimum pH (determined by changing the pH of buffer) of theenzyme of the present invention is shown in FIG. 4. The enzyme showedhigh activity at around pH 7.0-7.5. In this graph, the black circleshows the case when MES buffer was used and white circle shows the casewhen Tris-HCl buffer was used.

[0168] The pH stability of the enzyme of the present invention wasexamined in the same manner. FIG. 5 shows residual activity aftertreating the enzyme in each buffer at each pH, 4° C. for 5 hours. Theenzyme was comparatively stable at about pH 4-10, and particularlystable at pH 5-9. In this graph, the black triangle shows the case whenacetate buffer was used, the black circle shows the case when MES bufferwas used, the white circle shows the case when Tris-HCl buffer was used,and the white triangle shows the case when sodium hydrogencarbonatebuffer was used.

[0169] As shown in FIG. 6, the optimum temperature of the enzyme of thepresent invention was found to be at about 37° C. and the enzyme wasconsidered to retain sufficient activity in the range of 20-40° C. Thefrozen product was stable at −20° C. for at least several months.

[0170] The enzyme showed sufficient activity in the absence of divalentmetal ion. Inasmuch as it showed sufficient activity even in thepresence of 5 mM EDTA, which is a chelating agent, it is concluded thatthe enzyme does not require a divalent metal ion.

EXAMPLE 8

[0171] Determination of Amino Acid Sequence of Human α1-6Fucosyltransferase

[0172] Purified human α1-6 fucosyltransferase (1 μg) was subjected toSDS-polyacrylamide gel electrophoresis, after which the protein wastransferred to PVDF membrane (Millipore) by electroblotting. The PVDFmembrane was stained with Coomassie Brilliant Blue G250, and a singleband of α1-6 fucosyltransferase was detected at about 60 kDa. Then, thePVDF membrane containing said band was cut out, and, after destainingwith 50% methanol, subjected to Biosystem 473A protein sequencer(Applied Biosystems) to determine amino terminal amino acid sequence ofhuman α1-6 fucosyltransferase. The amino acid sequence determined isdepicted in Sequence Listing, SEQ ID NO:11.

EXAMPLE 9

[0173] Determination of Partial Amino Acid Sequence of Human α1-6Fucosyltransferase

[0174] Purified human α1-6 fucosyltransferase (5 μg) was mixed withlysine endopeptidase and subjected to SDS-polyacrylamide gelelectrophoresis, after which the peptide fragments were transferred toPVDF membrane (Millipore) by electroblotting. The PVDF membrane wasstained with Coomassie Brilliant Blue G250, and several bands containingpeptide fragments, inclusive of two main bands, were detected. Then, thePVDF membrane containing each main band was cut out and destained with50% methanol. Said membrane was subjected to Biosystem 473A proteinsequencer (Applied Biosystems) to determine the internal partial aminoacid sequence of human α1-6 fucosyltransferase. The determined aminoacid sequences are depicted in Sequence Listing, SEQ m NO:12 and SEQ IDNO:13.

EXAMPLE 10

[0175] Preparation of Probe DNA by PCR

[0176] Mixed primers shown by SEQ ID NO:14 and SEQ ID NO:15 weresynthesized from the amino acid sequences obtained in Example 9. Themixed primer shown in SEQ ID NO:14 was used as a sense primer, and themixed primer shown in SEQ ID NO:15 was used as an antisense primer forPCR. To be specific, 36 cycles of PCR were performed wherein PCR at 94°C. (30 sec), 46° C. (30 sec) and 72° C. (1.5 min) using 2 μg ofhuman-derived cDNA, 25 pmole of sense primer (mixed primer shown in SEQID NO: 14), 25 pmole of antisense primer (mixed primer shown in SEQ IDNO:15) and a reaction mixture (50 μl) of 50 mM potassium chloride-10 mMTris-HCl buffer (pH 8.3)-1.5 mM magnesium chloride-0.001% gelatin-200 μMdNTP, containing 2.5 units of Taq DNA polymerase, was one cycle.

[0177] The reaction mixture (10 μl) after PCR was subjected to 2.0%agarose gel electrophoresis to confirm the PCR reaction product DNAfragments. As a result, about 200 bp DNA fragment was confirmed byagarose gel electrophoresis.

[0178] This DNA fragment was suboloned into plasmid pT7BLUEt-Vector(Novagen) and the nucleotide sequence was confirmed. As a result, theDNA fragment was found to encode the amino acid sequence depicted inSequence Listing, SEQ ID NO:12 and SEQ ID NO:13, whereby the DNAfragment was confirmed to be a part of α1-6 fucosyltransferase gene.

EXAMPLE 11

[0179] Isolation of Human α1-6 Fucosyltransferase Gene

[0180] The DNA fragment obtained in Example 10 was labeled with[α-³²P]dCTP (3000 Ci/mmol, Amersham) and used as a probe to screenclones containing cDNA encoding human α1-6 fucosyltransferase, fromhuman gastric cancer cell MKN45-derived λ ZAP cDNA library by plaquehybridization method. As a result of screening of about 2,000,000plaques, 8 positive clones c1 to c8 were obtained. Said clones c1 to c7were postulated to contain a full length α1-6 fucosyltransferase gene inview of the restriction enzyme cleavage site and their length. Thenucleotide sequences of c1 and c2 were determined, as a result of whicha nucleotide sequence depicted in SEQ ID NO:9 was obtained.

EXAMPLE 12

[0181] Expression of Human α1-6 Fucosyltransferase

[0182] The coding region of human α1-6 fucosyltransferase gene wassubcloned into expression vector pSVK3 from clones containing cDNAencoding the human α1-6 fucosyltransferase obtained in Example 11. Anexpression vector containing said α1-6 fucosyltransferase gene wasintroduced into COS-1 cells. After 48 hours of incubation, culture cellswere collected and disrupted. The enzyme activity of α1-6fucosyltransferase in the obtained lysate was determined. As a control,the enzyme activity of α1-6 fucosyltransferase in the lysate of COS-1cells, into which mock pSVK3 had been introduced, was determined. As aresult, the control hardly showed activity, whereas COS-cells, intowhich the expression vector containing said α1-6 fucosyltransferase genehad been introduced, showed a high activity of 2130 nmole/h/mg protein.

INDUSTRIAL APPLICABILITY

[0183] The porcine α1-6 fucosyltransferase of the present inventiondiffers significantly from known human α1-6 fucosyltransferase inphysico-chemical properties, and shows activity under optimum reactionconditions which are closer to the physiological conditions.

[0184] The α1-6 fucosyltransferase derived from human also showsphysico-chemical properties markedly different from those of known humanα1-6 fucosyltransferase, showing activity under optimum reactionconditions which are closer to the physiological conditions. Hence, thepresent invention enables development of glyco-technology inclusive ofmodification and synthesis of sugar chain, and of a method for diagnosisof diseases, such as cancer, which includes the use of an antibodyspecific for the enzyme of the present invention or the gene thereof.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 15 <210> SEQ ID NO 1<211> LENGTH: 1728 <212> TYPE: DNA <213> ORGANISM: Pig <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(1728) <400> SEQUENCE: 1 atgcgg cca tgg act ggt tcg tgg cgt tgg att atg ctc att ctt ttt 48 Met ArgPro Trp Thr Gly Ser Trp Arg Trp Ile Met Leu Ile Leu Phe 1 5 10 15 gcctgg ggg acc ttg cta ttt tac ata ggt ggt cac ttg gta cga gat 96 Ala TrpGly Thr Leu Leu Phe Tyr Ile Gly Gly His Leu Val Arg Asp 20 25 30 aat gaccac tct gat cac tct agc cga gaa ctg tcc aag att ttg gca 144 Asn Asp HisSer Asp His Ser Ser Arg Glu Leu Ser Lys Ile Leu Ala 35 40 45 aag ctg gaacgc tta aaa caa caa aat gaa gac ttg agg aga atg gct 192 Lys Leu Glu ArgLeu Lys Gln Gln Asn Glu Asp Leu Arg Arg Met Ala 50 55 60 gga tct ctc cgaata cca gaa ggc ccc att gat cag ggg cca gct tca 240 Gly Ser Leu Arg IlePro Glu Gly Pro Ile Asp Gln Gly Pro Ala Ser 65 70 75 80 gga aga gtt cgtgct tta gaa gag caa ttt atg aag gcc aaa gaa cag 288 Gly Arg Val Arg AlaLeu Glu Glu Gln Phe Met Lys Ala Lys Glu Gln 85 90 95 att gaa aat tat aagaaa caa act aaa aat ggt cca ggg aag gat cat 336 Ile Glu Asn Tyr Lys LysGln Thr Lys Asn Gly Pro Gly Lys Asp His 100 105 110 gaa atc cta agg aggagg att gaa aat gga gct aaa gag ctc tgg ttt 384 Glu Ile Leu Arg Arg ArgIle Glu Asn Gly Ala Lys Glu Leu Trp Phe 115 120 125 ttt cta caa agt gagttg aag aaa tta aag aat tta gaa gga aat gaa 432 Phe Leu Gln Ser Glu LeuLys Lys Leu Lys Asn Leu Glu Gly Asn Glu 130 135 140 ctc caa aga cat gcagat gaa ttt cta tca gat ttg gga cat cat gaa 480 Leu Gln Arg His Ala AspGlu Phe Leu Ser Asp Leu Gly His His Glu 145 150 155 160 agg tct ata atgacg gat cta tac tac ctc agt caa aca gat ggg gca 528 Arg Ser Ile Met ThrAsp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala 165 170 175 ggt gat tgg cgtgaa aag gag gcc aaa gat ctg aca gag ctg gtc cag 576 Gly Asp Trp Arg GluLys Glu Ala Lys Asp Leu Thr Glu Leu Val Gln 180 185 190 cgg aga ata acatat ctt cag aat ccc aag gac tgc agc aaa gcc aag 624 Arg Arg Ile Thr TyrLeu Gln Asn Pro Lys Asp Cys Ser Lys Ala Lys 195 200 205 aag cta gtg tgtaat atc aac aaa ggc tgt ggc tat ggc tgt cag ctc 672 Lys Leu Val Cys AsnIle Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu 210 215 220 cat cat gta gtgtac tgc ttt atg att gca tat ggc acc cag cga aca 720 His His Val Val TyrCys Phe Met Ile Ala Tyr Gly Thr Gln Arg Thr 225 230 235 240 ctc gcc ttggaa tct cac aat tgg cgc tac gct act ggg gga tgg gaa 768 Leu Ala Leu GluSer His Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu 245 250 255 act gtg tttaga cct gta agt gag acg tgc aca gac aga tct ggc agc 816 Thr Val Phe ArgPro Val Ser Glu Thr Cys Thr Asp Arg Ser Gly Ser 260 265 270 tcc act ggacat tgg tca ggt gaa gta aag gac aaa aat gtt cag gtg 864 Ser Thr Gly HisTrp Ser Gly Glu Val Lys Asp Lys Asn Val Gln Val 275 280 285 gtt gag ctcccc att gta gac agt gtt cat cct cgt cct cca tat tta 912 Val Glu Leu ProIle Val Asp Ser Val His Pro Arg Pro Pro Tyr Leu 290 295 300 ccc ctg gctgtc cca gaa gac ctt gca gat cga ctt gta cga gtc cat 960 Pro Leu Ala ValPro Glu Asp Leu Ala Asp Arg Leu Val Arg Val His 305 310 315 320 ggt gatcct gca gtg tgg tgg gta tcc cag ttt gtc aag tac ttg att 1008 Gly Asp ProAla Val Trp Trp Val Ser Gln Phe Val Lys Tyr Leu Ile 325 330 335 cgc ccacaa ccc tgg ctg gaa aag gaa ata gaa gag gcc acc aag aag 1056 Arg Pro GlnPro Trp Leu Glu Lys Glu Ile Glu Glu Ala Thr Lys Lys 340 345 350 cta ggcttc aaa cat cca gtt att gga gtc cat gtt aga cgc aca gac 1104 Leu Gly PheLys His Pro Val Ile Gly Val His Val Arg Arg Thr Asp 355 360 365 aaa gtggga gcg gaa gca gcc ttc cat ccc att gag gaa tac acg gtg 1152 Lys Val GlyAla Glu Ala Ala Phe His Pro Ile Glu Glu Tyr Thr Val 370 375 380 cac gttgaa gaa gac ttt cag ctt ctt gct cgc aga atg caa gtg gat 1200 His Val GluGlu Asp Phe Gln Leu Leu Ala Arg Arg Met Gln Val Asp 385 390 395 400 aaaaaa agg gtg tat ttg gcc aca gat gac cct gct ttg tta aaa gag 1248 Lys LysArg Val Tyr Leu Ala Thr Asp Asp Pro Ala Leu Leu Lys Glu 405 410 415 gcaaaa aca aag tac ccc agt tat gaa ttt att agt gat aac tct atc 1296 Ala LysThr Lys Tyr Pro Ser Tyr Glu Phe Ile Ser Asp Asn Ser Ile 420 425 430 tcttgg tca gct gga cta cat aat cga tat aca gaa aat tca ctt cgg 1344 Ser TrpSer Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser Leu Arg 435 440 445 ggtgtg atc ctg gat ata cac ttt ctc tcc cag gca gac ttc cta gtg 1392 Gly ValIle Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val 450 455 460 tgtact ttt tca tcg cag gtc tgt aga gtt gct tat gaa atc atg caa 1440 Cys ThrPhe Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln 465 470 475 480gcg ctg cat cct gat gcc tct gcg aac ttc cgt tct ttg gat gac atc 1488 AlaLeu His Pro Asp Ala Ser Ala Asn Phe Arg Ser Leu Asp Asp Ile 485 490 495tac tat ttt gga ggc cca aat gcc cac aac caa att gcc att tat cct 1536 TyrTyr Phe Gly Gly Pro Asn Ala His Asn Gln Ile Ala Ile Tyr Pro 500 505 510cac caa cct cga act gaa gga gaa atc ccc atg gaa cct gga gat att 1584 HisGln Pro Arg Thr Glu Gly Glu Ile Pro Met Glu Pro Gly Asp Ile 515 520 525att ggt gtg gct gga aat cac tgg gat ggc tat cct aaa ggt gtt aac 1632 IleGly Val Ala Gly Asn His Trp Asp Gly Tyr Pro Lys Gly Val Asn 530 535 540aga aaa ctg gga agg acg ggc cta tat ccc tcc tac aaa gtt cga gag 1680 ArgLys Leu Gly Arg Thr Gly Leu Tyr Pro Ser Tyr Lys Val Arg Glu 545 550 555560 aag ata gaa aca gtc aag tac ccc aca tat ccc gag gct gac aag taa 1728Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala Asp Lys * 565 570575 <210> SEQ ID NO 2 <211> LENGTH: 575 <212> TYPE: PRT <213> ORGANISM:Pig <400> SEQUENCE: 2 Met Arg Pro Trp Thr Gly Ser Trp Arg Trp Ile MetLeu Ile Leu Phe 1 5 10 15 Ala Trp Gly Thr Leu Leu Phe Tyr Ile Gly GlyHis Leu Val Arg Asp 20 25 30 Asn Asp His Ser Asp His Ser Ser Arg Glu LeuSer Lys Ile Leu Ala 35 40 45 Lys Leu Glu Arg Leu Lys Gln Gln Asn Glu AspLeu Arg Arg Met Ala 50 55 60 Glu Ser Leu Arg Ile Pro Glu Gly Pro Ile AspGln Gly Pro Ala Ser 65 70 75 80 Gly Arg Val Arg Ala Leu Glu Glu Gln PheMet Lys Ala Lys Glu Gln 85 90 95 Ile Glu Asn Tyr Lys Lys Gln Thr Lys AsnGly Pro Gly Lys Asp His 100 105 110 Glu Ile Leu Arg Arg Arg Ile Glu AsnGly Ala Lys Glu Leu Trp Phe 115 120 125 Phe Leu Gln Ser Glu Leu Lys LysLeu Lys Asn Leu Glu Gly Asn Glu 130 135 140 Leu Gln Arg His Ala Asp GluPhe Leu Ser Asp Leu Gly His His Glu 145 150 155 160 Arg Ser Ile Met ThrAsp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala 165 170 175 Gly Asp Trp ArgGlu Lys Glu Ala Lys Asp Leu Thr Glu Leu Val Gln 180 185 190 Arg Arg IleThr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys Ala Lys 195 200 205 Lys LeuVal Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu 210 215 220 HisHis Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln Arg Thr 225 230 235240 Leu Ala Leu Glu Ser His Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu 245250 255 Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr Asp Arg Ser Gly Ser260 265 270 Ser Thr Gly His Trp Ser Gly Glu Val Lys Asp Lys Asn Val GlnVal 275 280 285 Val Glu Leu Pro Ile Val Asp Ser Val His Pro Arg Pro ProTyr Leu 290 295 300 Pro Leu Ala Val Pro Glu Asp Leu Ala Asp Arg Leu ValArg Val His 305 310 315 320 Gly Asp Pro Ala Val Trp Trp Val Ser Gln PheVal Lys Tyr Leu Ile 325 330 335 Arg Pro Gln Pro Trp Leu Glu Lys Glu IleGlu Glu Ala Thr Lys Lys 340 345 350 Leu Gly Phe Lys His Pro Val Ile GlyVal His Val Arg Arg Thr Asp 355 360 365 Lys Val Gly Ala Glu Ala Ala PheHis Pro Ile Glu Glu Tyr Thr Val 370 375 380 His Val Glu Glu Asp Phe GlnLeu Leu Ala Arg Arg Met Gln Val Asp 385 390 395 400 Lys Lys Arg Val TyrLeu Ala Thr Asp Asp Pro Ala Leu Leu Lys Glu 405 410 415 Ala Lys Thr LysTyr Pro Ser Tyr Glu Phe Ile Ser Asp Asn Ser Ile 420 425 430 Ser Trp SerAla Gly Leu His Asn Arg Tyr Thr Glu Asn Ser Leu Arg 435 440 445 Gly ValIle Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val 450 455 460 CysThr Phe Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln 465 470 475480 Ala Leu His Pro Asp Ala Ser Ala Asn Phe Arg Ser Leu Asp Asp Ile 485490 495 Tyr Tyr Phe Gly Gly Pro Asn Ala His Asn Gln Ile Ala Ile Tyr Pro500 505 510 His Gln Pro Arg Thr Glu Gly Glu Ile Pro Met Glu Pro Gly AspIle 515 520 525 Ile Gly Val Ala Gly Asn His Trp Asp Gly Tyr Pro Lys GlyVal Asn 530 535 540 Arg Lys Leu Gly Arg Thr Gly Leu Tyr Pro Ser Tyr LysVal Arg Glu 545 550 555 560 Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr ProGlu Ala Asp Lys 565 570 575 <210> SEQ ID NO 3 <211> LENGTH: 26 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer <400> SEQUENCE: 3 Lys Gln Thr Lys Asn Gly Pro GlyLys Asp His Glu Ile Leu Arg Arg 1 5 10 15 Arg Ile Glu Asn Gly Ala LysGlu Leu Gln 20 25 <210> SEQ ID NO 4 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer <400> SEQUENCE: 4 Lys Tyr Pro Thr Tyr Pro Glu AlaAsp Lys 1 5 10 <210> SEQ ID NO 5 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Primer <400> SEQUENCE: 5 Lys Tyr Leu Ile Arg Pro Gln Pro Trp Leu Glu Lys1 5 10 <210> SEQ ID NO 6 <211> LENGTH: 14 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Primer <400> SEQUENCE: 6 Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro Ala LeuLeu Lys 1 5 10 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Primer <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 9, 18<223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: 9, 18 <223> OTHER INFORMATION: n = A,T,C orG <400> SEQUENCE: 7 aarsaracna araayggncc 20 <210> SEQ ID NO 8 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Primer <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: 3, 9, 12 <223> OTHER INFORMATION: n = A,T,Cor G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 3, 9,12 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 8 tcnggrtangtnggrtaytt 20 <210> SEQ ID NO 9 <211> LENGTH: 2100 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (198)...(1925) <400> SEQUENCE: 9 aagcttccta cacatatcaccaggaggatc tctttgaaag attcactgca ggactaccag 60 agagaataat ttgtctgaagcatcatgtgt tgaaacaaca gaagtctatt cacctgtgca 120 ctaactagaa acagagttacaatgttttca attctttgag ctccaggact ccagggaagt 180 gagttgaaaa tctgaaa atgcgg cca tgg act ggt tcc tgg cgt tgg att 230 Met Arg Pro Trp Thr Gly SerTrp Arg Trp Ile 1 5 10 atg ctc att ctt ttt gcc tgg ggg acc ttg ctg ttttat ata ggt ggt 278 Met Leu Ile Leu Phe Ala Trp Gly Thr Leu Leu Phe TyrIle Gly Gly 15 20 25 cac ttg gta cga gat aat gac cat cct gat cac tct agccga gaa ctg 326 His Leu Val Arg Asp Asn Asp His Pro Asp His Ser Ser ArgGlu Leu 30 35 40 tcc aag att ctg gca aag ctt gaa cgc tta aaa cag cag aatgaa gac 374 Ser Lys Ile Leu Ala Lys Leu Glu Arg Leu Lys Gln Gln Asn GluAsp 45 50 55 ttg agg cga atg gcc gaa tct ctc cgg ata cca gaa ggc cct attgat 422 Leu Arg Arg Met Ala Glu Ser Leu Arg Ile Pro Glu Gly Pro Ile Asp60 65 70 75 cag ggg cca gct ata gga aga gta cgc gtt tta gaa gag cag cttgtt 470 Gln Gly Pro Ala Ile Gly Arg Val Arg Val Leu Glu Glu Gln Leu Val80 85 90 aag gcc aaa gaa cag att gaa aat tac aag aaa cag acc aga aat ggt518 Lys Ala Lys Glu Gln Ile Glu Asn Tyr Lys Lys Gln Thr Arg Asn Gly 95100 105 ctg ggg aag gat cat gaa atc ctg agg agg agg att gaa aat gga gct566 Leu Gly Lys Asp His Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala 110115 120 aaa gag ctc tgg ttt ttc cta cag agt gaa ttg aag aaa tta aag aac614 Lys Glu Leu Trp Phe Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys Asn 125130 135 tta gaa gga aat gaa ctc caa aga cat gca gat gaa ttt ctt ttg gat662 Leu Glu Gly Asn Glu Leu Gln Arg His Ala Asp Glu Phe Leu Leu Asp 140145 150 155 tta gga cat cat gaa agg tct ata atg acg gat cta tac tac ctcagt 710 Leu Gly His His Glu Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu Ser160 165 170 cag aca gat gga gca ggt gat tgg cgg gaa aaa gag gcc aaa gatctg 758 Gln Thr Asp Gly Ala Gly Asp Trp Arg Glu Lys Glu Ala Lys Asp Leu175 180 185 aca gaa ctg gtt cag cgg aga ata aca tat ctt cag aat ccc aaggac 806 Thr Glu Leu Val Gln Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp190 195 200 tgc agc aaa gcc aaa aag ctg gtg tgt aat atc aac aaa ggc tgtggc 854 Cys Ser Lys Ala Lys Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly205 210 215 tat ggc tgt cag ctc cat cat gtg gtc tac tgc ttc atg att gcatat 902 Tyr Gly Cys Gln Leu His His Val Val Tyr Cys Phe Met Ile Ala Tyr220 225 230 235 ggc acc cag cga aca ctc atc ttg gaa tct cag aat tgg cgctat gct 950 Gly Thr Gln Arg Thr Leu Ile Leu Glu Ser Gln Asn Trp Arg TyrAla 240 245 250 act ggt gga tgg gag act gta ttt agg cct gta agt gag acatgc aca 998 Thr Gly Gly Trp Glu Thr Val Phe Arg Pro Val Ser Glu Thr CysThr 255 260 265 gac aga tct ggc atc tcc act gga cac tgg tca ggt gaa gtgaag gac 1046 Asp Arg Ser Gly Ile Ser Thr Gly His Trp Ser Gly Glu Val LysAsp 270 275 280 aaa aat gtt caa gtg gtc gag ctt ccc att gta gac agt cttcat ccc 1094 Lys Asn Val Gln Val Val Glu Leu Pro Ile Val Asp Ser Leu HisPro 285 290 295 cgt cct cca tat tta ccc ttg gct gta cca gaa gac ctc gcagat cga 1142 Arg Pro Pro Tyr Leu Pro Leu Ala Val Pro Glu Asp Leu Ala AspArg 300 305 310 315 ctt gta cga gtg cat ggt gac cct gca gtg tgg tgg gtgtct cag ttt 1190 Leu Val Arg Val His Gly Asp Pro Ala Val Trp Trp Val SerGln Phe 320 325 330 gtc aaa tac ttg atc cgc cca cag cct tgg cta gaa aaagaa ata gaa 1238 Val Lys Tyr Leu Ile Arg Pro Gln Pro Trp Leu Glu Lys GluIle Glu 335 340 345 gaa gcc acc aag aag ctt ggc ttc aaa cat cca gtt attgga gtc cat 1286 Glu Ala Thr Lys Lys Leu Gly Phe Lys His Pro Val Ile GlyVal His 350 355 360 gtc aga cgc aca gac aaa gtg gga aca gaa gct gcc ttccat ccc att 1334 Val Arg Arg Thr Asp Lys Val Gly Thr Glu Ala Ala Phe HisPro Ile 365 370 375 gaa gag tac atg gtg cat gtt gaa gaa cat ttt cag cttctt gca cgc 1382 Glu Glu Tyr Met Val His Val Glu Glu His Phe Gln Leu LeuAla Arg 380 385 390 395 aga atg caa gtg gac aaa aaa aga gtg tat ttg gccaca gat gac cct 1430 Arg Met Gln Val Asp Lys Lys Arg Val Tyr Leu Ala ThrAsp Asp Pro 400 405 410 tct tta tta aag gag gca aaa aca aag tac ccc aattat gaa ttt att 1478 Ser Leu Leu Lys Glu Ala Lys Thr Lys Tyr Pro Asn TyrGlu Phe Ile 415 420 425 agt gat aac tct att tcc tgg tca gct gga ctg cacaat cga tac aca 1526 Ser Asp Asn Ser Ile Ser Trp Ser Ala Gly Leu His AsnArg Tyr Thr 430 435 440 gaa aat tca ctt cgt gga gtg atc ctg gat ata catttt ctc tct cag 1574 Glu Asn Ser Leu Arg Gly Val Ile Leu Asp Ile His PheLeu Ser Gln 445 450 455 gca gac ttc cta gtg tgt act ttt tca tcc cag gtctgt cga gtt act 1622 Ala Asp Phe Leu Val Cys Thr Phe Ser Ser Gln Val CysArg Val Thr 460 465 470 475 tat gaa att atg caa aca cta cat cct gat gcctct gca aac ttc cat 1670 Tyr Glu Ile Met Gln Thr Leu His Pro Asp Ala SerAla Asn Phe His 480 485 490 tct tta gat gac atc tac tat ttt ggg ggc cagaat gcc cac aat caa 1718 Ser Leu Asp Asp Ile Tyr Tyr Phe Gly Gly Gln AsnAla His Asn Gln 495 500 505 att gcc att tat gct cac caa ccc cga act gcagat gaa att ccc atg 1766 Ile Ala Ile Tyr Ala His Gln Pro Arg Thr Ala AspGlu Ile Pro Met 510 515 520 gaa cct gga gat atc att ggt gtg gct gga aatcat tgg gat ggc tat 1814 Glu Pro Gly Asp Ile Ile Gly Val Ala Gly Asn HisTrp Asp Gly Tyr 525 530 535 tct aaa ggt gtc aac agg aaa ttg gga agg acgggc cta tat ccc tcc 1862 Ser Lys Gly Val Asn Arg Lys Leu Gly Arg Thr GlyLeu Tyr Pro Ser 540 545 550 555 tac aaa gtt cca gag aag ata gaa acg gtcaag tac ccc aca tat cct 1910 Tyr Lys Val Pro Glu Lys Ile Glu Thr Val LysTyr Pro Thr Tyr Pro 560 565 570 gag gct gag aaa taa agctcacatggaagagataa acgaccaaac tcagttcgac 1965 Glu Ala Glu Lys * 575 caaactcagttcaaaccatt tcagccaaac tgtagatgaa gagggctctg atctaacaaa 2025 ataaggttatatgagtagat actctcagca ccaagagcag ctgggaactg acataggctt 2085 caattggtggaattc 2100 <210> SEQ ID NO 10 <211> LENGTH: 575 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 10 Met Arg Pro Trp Thr Gly SerTrp Arg Trp Ile Met Leu Ile Leu Phe 1 5 10 15 Ala Trp Gly Thr Leu LeuPhe Tyr Ile Gly Gly His Leu Val Arg Asp 20 25 30 Asn Asp His Pro Asp HisSer Ser Arg Glu Leu Ser Lys Ile Leu Ala 35 40 45 Lys Leu Glu Arg Leu LysGln Gln Asn Glu Asp Leu Arg Arg Met Ala 50 55 60 Glu Ser Leu Arg Ile ProGlu Gly Pro Ile Asp Gln Gly Pro Ala Ile 65 70 75 80 Gly Arg Val Arg ValLeu Glu Glu Gln Leu Val Lys Ala Lys Glu Gln 85 90 95 Ile Glu Asn Tyr LysLys Gln Thr Arg Asn Gly Leu Gly Lys Asp His 100 105 110 Glu Ile Leu ArgArg Arg Ile Glu Asn Gly Ala Lys Glu Leu Trp Phe 115 120 125 Phe Leu GlnSer Glu Leu Lys Lys Leu Lys Asn Leu Glu Gly Asn Glu 130 135 140 Leu GlnArg His Ala Asp Glu Phe Leu Leu Asp Leu Gly His His Glu 145 150 155 160Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala 165 170175 Gly Asp Trp Arg Glu Lys Glu Ala Lys Asp Leu Thr Glu Leu Val Gln 180185 190 Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys Ala Lys195 200 205 Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys GlnLeu 210 215 220 His His Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr GlnArg Thr 225 230 235 240 Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala ThrGly Gly Trp Glu 245 250 255 Thr Val Phe Arg Pro Val Ser Glu Thr Cys ThrAsp Arg Ser Gly Ile 260 265 270 Ser Thr Gly His Trp Ser Gly Glu Val LysAsp Lys Asn Val Gln Val 275 280 285 Val Glu Leu Pro Ile Val Asp Ser LeuHis Pro Arg Pro Pro Tyr Leu 290 295 300 Pro Leu Ala Val Pro Glu Asp LeuAla Asp Arg Leu Val Arg Val His 305 310 315 320 Gly Asp Pro Ala Val TrpTrp Val Ser Gln Phe Val Lys Tyr Leu Ile 325 330 335 Arg Pro Gln Pro TrpLeu Glu Lys Glu Ile Glu Glu Ala Thr Lys Lys 340 345 350 Leu Gly Phe LysHis Pro Val Ile Gly Val His Val Arg Arg Thr Asp 355 360 365 Lys Val GlyThr Glu Ala Ala Phe His Pro Ile Glu Glu Tyr Met Val 370 375 380 His ValGlu Glu His Phe Gln Leu Leu Ala Arg Arg Met Gln Val Asp 385 390 395 400Lys Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro Ser Leu Leu Lys Glu 405 410415 Ala Lys Thr Lys Tyr Pro Asn Tyr Glu Phe Ile Ser Asp Asn Ser Ile 420425 430 Ser Trp Ser Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser Leu Arg435 440 445 Gly Val Ile Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe LeuVal 450 455 460 Cys Thr Phe Ser Ser Gln Val Cys Arg Val Thr Tyr Glu IleMet Gln 465 470 475 480 Thr Leu His Pro Asp Ala Ser Ala Asn Phe His SerLeu Asp Asp Ile 485 490 495 Tyr Tyr Phe Gly Gly Gln Asn Ala His Asn GlnIle Ala Ile Tyr Ala 500 505 510 His Gln Pro Arg Thr Ala Asp Glu Ile ProMet Glu Pro Gly Asp Ile 515 520 525 Ile Gly Val Ala Gly Asn His Trp AspGly Tyr Ser Lys Gly Val Asn 530 535 540 Arg Lys Leu Gly Arg Thr Gly LeuTyr Pro Ser Tyr Lys Val Pro Glu 545 550 555 560 Lys Ile Glu Thr Val LysTyr Pro Thr Tyr Pro Glu Ala Glu Lys 565 570 575 <210> SEQ ID NO 11 <211>LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 11 Arg Ile ProGlu Gly Pro Ile Asp Gln Gly Pro Ala Ile Gly 1 5 10 <210> SEQ ID NO 12<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 12 LysLeu Gly Phe Lys His Pro Val Ile Gly Val His Val Arg Arg Thr 1 5 10 15Asp Lys Val Gly Thr Cys Ala Ala Phe 20 25 <210> SEQ ID NO 13 <211>LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: 223> OTHER INFORMATION: Primer <400> SEQUENCE: 13 Thr Lys TyrPro Asn Tyr Glu Phe Ile Ser Asp Asn Ser 1 5 10 <210> SEQ ID NO 14 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 14 ttyaarcaycchgtbatygg 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Primer <400> SEQUENCE: 15 gwrttrtcrg wratraaytc 20

1-26. (canceled).
 27. An isolated polynucleotide encoding amino acidsequence as depicted in Sequence Listing, SEQ ID NO:2.
 28. The isolatedpolynucleotide of claim 27, comprising a nucleotide sequence as depictedin Sequence Listing, SEQ ID NO:1.
 29. An expression vector whichcomprises the isolated polynucleotide of claim
 27. 30. An expressionvector which comprises the isolated polynucleotide of claim
 28. 31. Atransformant cell obtained by transforming a host cell with theexpression vector of claim
 29. 32. A transformant cell obtained bytransforming a host cell with the expression vector of claim 30
 33. Amethod for producing a recombinant α1-6 fucosyltransferase, comprisingculturing the transformant cell of claim 31, and harvesting the α1-6fucosyltransferase from a culture thereof.
 34. A method for producing arecombinant α1-6 fucosyltransferase, comprising culturing thetransformant cell of claim 32, and harvesting the α1-6fucosyltransferase from a culture thereof.
 35. An isolatedpolynucleotide encoding α1-6 fucosyltransferase derived from porcinetissue, having the following physico-chemical properties: (1) action:transferring fucose from guanosine diphosphate-fucose to a hydroxy groupat 6-position of GluNAc closest to R of a receptor (GlcNAc1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manα1-4GlcNAcβ1-4GlucNAc-R wherein R is anasparagine residue or a peptide chain carrying said residue, whereby toform(GlcNAcβ1-2Manα1-6)-(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlucNAc-R(2) optimum pH: about 7.0 (3) pH stability: stable in the pH range of4.0-10.0 by treatment at 4° C. for 5 hours (4) optimum temperature:about 30-37° C. (5) inhibition or activation: no requirement fordivalent metal for expression of activity; no inhibition of activity inthe presence of 5 mM EDTA (6) molecular weight: about 60,000 bySDS-polyacrylamide gel electrophoresis.
 36. An expression vector whichcomprises the isolated polynucleotide of claim 35.